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Lactoferricin Peptides IncreaseMacrophages’ Capacity To
KillMycobacterium avium
Tânia Silva,a,b,c,d Ana C. Moreira,a,b Kamran Nazmi,e Tânia
Moniz,f Nuno Vale,f
Maria Rangel,d,f Paula Gomes,f Jan G. M. Bolscher,e Pedro N.
Rodrigues,a,b,d
Margarida Bastos,c Maria Salomé Gomesa,b,d
i3S, Instituto de Investigação e Inovação em Saúde, Universidade
do Porto, Porto, Portugala; Instituto deBiologia Molecular e
Celular (IBMC), Universidade do Porto, Porto, Portugalb; Centro de
Investigação emQuímica, Departamento de Química e Bioquímica,
Faculdade de Ciências, Universidade do Porto, Porto,Portugalc;
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade
do Porto, Porto, Portugald;Department of Oral Biochemistry,
Academic Centre for Dentistry Amsterdam (ACTA), University of
Amsterdam,and VU University Amsterdam, Amsterdam, The Netherlandse;
REQUIMTE-UCIBIO, Departamento de Química eBioquímica, Faculdade de
Ciências, Universidade do Porto, Porto, Portugalf
ABSTRACT Mycobacterial infections cause a significant burden of
disease anddeath worldwide. Their treatment is long, toxic, costly,
and increasingly prone to fail-ure due to bacterial resistance to
currently available antibiotics. New therapeutic op-tions are thus
clearly needed. Antimicrobial peptides represent an important
sourceof new antimicrobial molecules, both for their direct
activity and for their immuno-modulatory potential. We have
previously reported that a short version of the bo-vine
antimicrobial peptide lactoferricin with amino acids 17 to 30
(LFcin17–30),along with its variants obtained by specific amino
acid substitutions, killed Mycobac-terium avium in broth culture.
In the present work, those peptides were testedagainst M. avium
living inside its natural host cell, the macrophage. We found
thatthe peptides increased the antimicrobial action of the
conventional antibiotic etham-butol inside macrophages. Moreover,
the D-enantiomer of the lactoferricin peptide(D-LFcin17–30) was
more stable and induced significant killing of intracellular
myco-bacteria by itself. Interestingly, D-LFcin17–30 did not
localize to M. avium-harboringphagosomes but induced the production
of proinflammatory cytokines and in-creased the formation of
lysosomes and autophagosome-like vesicles. These resultslead us to
conclude that D-LFcin17–30 primes macrophages for intracellular
micro-bial digestion through phagosomal maturation and/or
autophagy, culminating inmycobacterial killing.
IMPORTANCE The genus Mycobacterium comprises several pathogenic
species, in-cluding M. tuberculosis, M. leprae, M. avium, etc.
Infections caused by these bacteriaare particularly difficult to
treat due to their intrinsic impermeability, low growthrate, and
intracellular localization. Antimicrobial peptides are increasingly
acknowl-edged as potential treatment tools, as they have a high
spectrum of activity, lowtendency to induce bacterial resistance,
and immunomodulatory properties. In thisstudy, we show that
peptides derived from bovine lactoferricin (LFcin) improve
theantimicrobial activity of ethambutol against Mycobacterium avium
growing insidemacrophages. Moreover, the D-enantiomer of a short
version of lactoferricin contain-ing amino acids 17 to 30
(D-LFcin17–30) causes intramacrophagic death of M. aviumby
increasing the formation of lysosomes and autophagosomes. This work
opens theway to the use of lactoferricin-derived peptides to treat
infections caused by myco-bacteria and highlights important
modulatory effects of D-FLcin17–30 on macro-phages, which may be
useful under other conditions in which macrophage activa-tion is
needed.
Received 6 July 2017 Accepted 4 August2017 Published 30 August
2017
Citation Silva T, Moreira AC, Nazmi K, Moniz T,Vale N, Rangel M,
Gomes P, Bolscher JGM,Rodrigues PN, Bastos M, Gomes MS.
2017.Lactoferricin peptides increase macrophages'capacity to kill
Mycobacterium avium. mSphere2:e00301-17.
https://doi.org/10.1128/mSphere.00301-17.
Editor Paul Dunman, University of Rochester
Copyright © 2017 Silva et al. This is an open-access article
distributed under the terms ofthe Creative Commons Attribution
4.0International license.
Address correspondence to Maria SaloméGomes,
[email protected].
RESEARCH ARTICLEHost-Microbe Biology
crossm
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KEYWORDS autophagy, lactoferricin, Mycobacterium, antimicrobial
peptide,macrophage
The Mycobacterium genus contains several species capable of
causing severe dis-ease, such as those belonging to the M.
tuberculosis complex, M. leprae, andnontuberculous mycobacteria
(NTM) (1, 2). The incidence of NTM infections, predom-inantly by
species of the M. avium complex (MAC), is increasing worldwide,
surpassingin some regions the number of infections caused by M.
tuberculosis (3, 4). Disseminatedinfections caused by NTM occur
mainly in patients with a compromised immunesystem, such as
HIV-infected patients, patients with cancer, and organ or stem
celltransplant patients, among others (reviewed in references 2, 5,
and 6).
Mycobacteria are characterized by a unique, complex, highly
impermeable cell walland are able to proliferate inside phagocytic
cells, subverting the intracellular vesiculartrafficking. These
characteristics confer upon them high resistance to chemotherapyand
the ability to cause persistent infections (7, 8). Treatment
regimens are based on acombination of several drugs taken for
months to years and in general have limitedefficacies (9, 10).
Furthermore, mycobacterial antibiotic resistance is increasing
world-wide, urging the need to develop novel classes of
antimicrobial drugs (11).
Mycobacteria are facultative intracellular pathogens residing
mainly inside macro-phages. After being phagocytized, the
mycobacteria arrest the maturation of thephagosome, inhibiting the
phagosome-lysosome fusion (12–14). This inhibition
enablesmycobacteria not only to escape the harmful environment of
lysosomes but also tomaintain the interaction with endosomes in the
recycling pathway, allowing theiraccess to nutrients [e.g.,
transferrin-bound Fe(III)] needed to ensure survival
andproliferation inside the host (12, 15, 16). Cytokines such as
gamma interferon (IFN-�)and tumor necrosis factor alpha (TNF-�)
play an important role in macrophage activa-tion and mycobacterial
growth restriction (17–20). However, the mechanisms by
whichmacrophages inhibit mycobacterial growth and the mechanisms
used by mycobacteriato resist and live inside macrophages are not
fully understood. Respiratory burst andnitric oxide (NO) are
involved in M. tuberculosis killing (21–23), but they do not seem
toplay an important role in the case of M. avium (15, 24, 25).
Nutrient restriction, includingthat of iron, is also thought to
have a role, namely, through alterations in vesiculartrafficking
that affect mycobacterium-harboring phagosomes (15). Cell death
mecha-nisms are also important for cell homeostasis and infection
control. In fact, mycobac-teria are known to modulate pathways such
as apoptosis, autophagy, necrosis, andpyroptosis, which have been
implicated in infection containment but also in enhancedbacterial
spread (26–28).
Antimicrobial peptides (AMP) are an important component of the
innate immuneresponse against pathogens. These peptides are
widespread in nature as part of hostdefense mechanisms,
constituting potential new antimicrobial treatment options
(29).Although their mode of action is still under debate, they are
thought to act with amultiple-hit strategy, which probably
contributes to their high efficacy and largespectrum of activity.
AMP can act directly on pathogens, either by disrupting themembrane
due to pore formation and/or micellization or by acting on internal
targets(30). They can also act by immunomodulation, being involved
in several processes, suchas modulation of pro- and
anti-inflammatory responses, chemoattraction,
cellulardifferentiation, angiogenesis, wound healing, enhancement
of bacterial clearance,autophagy, and apoptosis, among others (31).
In the case of mycobacteria, one of themost effective mechanisms of
host resistance is the vitamin D-dependent induction ofan AMP
(LL-37) and autophagy (32–34).
Lactoferricin is an antimicrobial peptide obtained by pepsin
digestion of the highlycationic N1 terminal domain of the
iron-binding protein lactoferrin (35, 36). The bovinelactoferricin
is composed of 25 amino acids (positions 17 to 41 in the native
protein)(37) and has a broad-spectrum antimicrobial activity
(reviewed in reference 38). Ashorter version, with amino acids 17
to 30 (LFcin17–30), was found to have high
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antimicrobial activity against both Gram-positive and
Gram-negative bacteria (39). Wehave previously shown that arginine
residues are crucial for the antimicrobial activity ofLFcin17–30
against M. avium growing in broth culture and that the
D-enantiomer(D-LFcin17–30) was even more active than the
L-enantiomer (40). In the present work,LFcin17–30 and its variants
were tested against M. avium growing inside mousemacrophages, alone
or in combination with the conventional antibiotic ethambutol.We
found that the D-LFcin17–30 enantiomer was the most active peptide,
actingthrough modulation of macrophages’ defense mechanisms.
RESULTSUp to 40 �M, lactoferricin peptides are not toxic to
primary mouse macro-
phages. Previously (40), we showed that bovine LFcin17–30 and
its variants with allarginines replaced with lysines and vice versa
(LFcin17–30 all K and LFcin17–30 all R,respectively), as well as
the variant with all amino acids in the D-form (D-LFcin17–30)(Table
1), killed M. avium in axenic cultures. In this work, we decided to
investigatewhether those peptides were able to kill mycobacteria
growing inside macrophages,their natural host cells. We have also
tested the possible synergistic effect of lactoferri-cin peptides
with ethambutol, a conventional antibiotic used in the clinics to
treatmycobacterial infections (41, 42). Before testing the
compounds for their antimicrobialactivity, we evaluated their
potential toxicity toward bone marrow-derived macro-phages (BMM).
In Fig. 1A and B, we show that the peptides, alone or in
combinationwith ethambutol, did not exert a significant toxic
effect on noninfected (data notshown) or infected macrophages at 40
�M, 1 and 5 days after incubation.
Lactoferricin peptides inhibit M. avium growth inside
macrophages and syn-ergize with ethambutol. Given that the peptides
at up to 40 �M were not toxic tomacrophages, we evaluated their
effect on M. avium growing inside these cells. Bonemarrow-derived
macrophages were obtained from BALB/c mice and infected with
M.avium 2447 smooth transparent variant (SmT). The different
peptides were added at 40�M, and ethambutol was added at 7.2 �M.
After 5 days in culture, the number ofintracellular bacteria per
culture well was quantified in a CFU assay (Fig. 1D). Among
thepeptides tested, only D-LFcin17–30 significantly inhibited the
intramacrophagic growthof M. avium (52% growth reduction, P �
0.001). None of the other peptides orethambutol alone significantly
inhibited M. avium growth. Interestingly, when given tothe
macrophages in combination with ethambutol, all peptides had a
significantinhibitory effect, revealing a possible synergistic
effect between antibiotic and AMP. Ofnote, even in combination with
ethambutol, D-LFcin17–30 was still the most activepeptide (73%
reduction in M. avium growth relative to the control; P �
0.001).
D-LFcin17–30 is more resistant to degradation by medium
components thanLFcin17–30. To understand the reason why
D-LFcin17–30 had a stronger effect on theintramacrophagic growth of
M. avium than LFcin17–30, and considering that peptidedegradation
is one of the factors that can have an impact on efficacy, we
evaluated byhigh-performance liquid chromatography (HPLC) the
kinetics of degradation of bothpeptides in the presence of the cell
culture medium used in the infection assays. Asexpected, the
peptide composed of amino acids in the D-form was significantly
moreresistant to degradation, persisting with no more than 30%
degradation for up to 96 hof incubation, whereas 50% of the L-form
of the peptide was degraded after 24 h ofincubation, being
completely degraded after 96 h (Fig. 1C).
TABLE 1 Characteristics of synthetic lactoferricin peptides
Peptide Amino acid sequence Molecular wt Chargea
LFcin17–30 FKCRRWQWRMKKLG 1,923 �6D-LFcin17–30 FKCRRWQWRMKKLG
1,923 �6LFcin17–30 all K FKCKKWQWKMKKLG 1,839 �6LFcin17–30 all R
FRCRRWQWRMRRLG 2,007 �6aCalculated overall charge at a pH of
7.0.
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Lactoferricin peptides do not colocalize with M. avium inside
macrophages. Inorder to understand the mechanisms by which
lactoferricin peptides inhibited theintramacrophagic growth of M.
avium, we characterized the intracellular distribution ofthe
peptides inside M. avium-infected macrophages. For that, we used
peptides labeledwith TAMRA [5(6)-carboxytetramethylrhodamine, a
rhodamine derivative], a strain of M.avium expressing green
fluorescent protein (GFP), and fluorescein-labeled markers
ofendosomes or mitochondria. Figure 2 depicts representative
pictures of macrophages2 h after infection with M. avium and
peptide treatment. LFcin17–30 (Fig. 2A) andD-LFcin17–30 (Fig. 2B)
exhibited similar distributions inside macrophages, and
neithercolocalized with M. avium. The exclusion of the peptides
from mycobacterium-containing vesicles was not altered by the
treatment with ethambutol (Fig. 2A and B,second column), by the
incubation time (20 min for up to 24 h [data not shown]), or
the
FIG 1 Effect of lactoferricin peptides on M. avium-infected
macrophages. (A) BALB/c mouse BMM were infected with M. avium
2447SmT and incubated with LFcin17–30 (gray circles), D-LFcin17–30
(black squares), LFcin17–30 all K (white triangles), and LFcin17–30
allR (white diamonds) for 24 h. At the end of this period, 10%
resazurin (125 �M) was added, and 24 h later fluorescence was
measuredat 560/590 nm to evaluate cell viability. The graph shows
the averages � standard deviations of results of two
independentexperiments, presented as percentages of viable cells
relative to the number of corresponding non-peptide-treated
infected cells. (B)BALB/c BMM were infected with M. avium 2447 SmT
and treated with 40 �M LFcin17–30, D-LFcin17–30, LFcin17–30 all K,
or LFcin17–30all R alone (nonpatterned bars) or in combination with
7.2 �M ethambutol (patterned bars). After 5 days of incubation, 10%
resazurin(125 �M) was added, and 24 h later fluorescence was
measured at 560/590 nm to evaluate cell viability. The graph shows
theaverages � standard deviations of results from three independent
experiments, presented as percentages of viable cells relative
tothe number of corresponding non-peptide-treated infected cells.
(C) LFcin17–30 (gray circles) and D-LFcin17–30 (black squares) at
a40 �M final concentration were incubated with cell medium at 37°C.
After 0, 0.5, 2, 4, 8, 24, 48, 72, and 96 h of incubation, a
40-�laliquot was immediately injected for RP-HPLC analysis using an
elution gradient of 0 to 100% acetonitrile in 0.05%
aqueoustrifluoroacetic acid (TFA) for 30 min at a flow rate of 1
ml/min. The results are presented as percentages of the remaining
peptide inrelation to the amount of peptide present at time zero.
(D) BALB/c BMM were infected with M. avium 2447 SmT and treated
with 40�M LFcin17–30, D-LFcin17–30, LFcin17–30 all K, or LFcin17–30
all R alone (nonpatterned bars) or in combination with 7.2
�Methambutol (patterned bars). After 5 days of incubation, bacteria
were quantified by a CFU assay. The results represent the averages
�standard deviations from at least four independent experiments and
are expressed as the percentage of intramacrophagic myco-bacteria
in each well relative to the number of mycobacteria in the
nontreated infected cells (control) in each experiment.
Statisticswere performed using two-way ANOVA with Tukey’s
multiple-comparison test. *, P � 0.05; **, P � 0.01; ***, P � 0.001
compared tonontreated wells (control); #, P � 0.001 compared to
ethambutol alone.
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time of peptide addition, either immediately after infection
(Fig. 2) or 4 to 5 days afterinfection (data not shown). Because
the intracellular distribution of both peptides hada vesicular
appearance, we studied their colocalization with the endocytic
pathway. Forthat, M. avium-infected macrophages were coincubated
with peptides and dextran-fluorescein isothiocyanate (FITC) for 2
h, and we found that both peptides extensively
FIG 2 Intracellular distribution and localization of
lactoferricin peptides in M. avium-infected macrophages. Thefigure
shows live-cell imaging of BALB/c BMM infected with M. avium and
treated with 10 �M red fluorescentpeptide for 2 h: LFcin17–30
—TAMRA (A) or D-LFcin17–30 —TAMRA (B). First column, M.
avium-GFP-infectedmacrophages; second column, M. avium-GFP-infected
macrophages treated with 7.2 �M ethambutol for 2 h; thirdcolumn, M.
avium 2447 SmT-infected macrophages incubated with 22.5 �M
fluorescein-conjugated dextran for 2h; forth column, M. avium 2447
SmT-infected macrophages incubated with 200 nM MitoTracker Green
for 30 min.One representative cell of one representative experiment
out of three is shown for each condition. Scale bar, 5 �m.
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colocalized with endosomes (Fig. 2A and B, third column),
suggesting that they areinternalized by this pathway. Importantly,
neither LFcin17–30 nor D-LFcin17–30 signif-icantly localized with
mitochondria, which indicates that they will not exert a
toxiceffect on this organelle (Fig. 2A and B, fourth column). The
evaluation of macrophageviability by resazurin reduction also
indicated that the TAMRA-labeled peptides had notoxicity toward the
macrophages under the conditions of the assay (data not shown).
Lactoferricin peptides increase macrophage production of
proinflammatorycytokines. Considering that lactoferricin peptides
appeared to decrease M. aviumviability inside macrophages without a
direct interaction with the bacteria (Fig. 2), wequestioned whether
they had a modulatory effect on macrophage function. For that,we
used macrophage supernatants to measure the levels of several
cytokines 24 h afterinfection with M. avium and concomitant
treatment with the peptides. The treatmentwith lactoferricin
peptides significantly increased the production of interleukin 6
(IL-6)(Fig. 3A) and TNF-� (Fig. 3B) by BMM infected with M. avium
(but not by noninfectedmacrophages [data not shown]), with no
significant differences between the twopeptides. IL-1�, IL-10,
CCL2, IL-12p40, and IFN-� were not significantly induced eitherby
M. avium infection or by peptide treatments (data not shown).
The antimicrobial effects of lactoferricin peptides inside
macrophages are notdependent on the production of TNF-� and/or of
IL-6 by macrophages. Bothpeptides increased the production of TNF-�
by M. avium-infected macrophages, andmacrophage activation by TNF-�
can lead to intracellular killing of mycobacteria (19,20);
therefore, we tested whether this cytokine was necessary for the
antibacterial effectof the peptides. We took BMM from Tnf�/� mice
and from congenic C57BL/6 wild-typemice, infected them with M.
avium 2447 SmT, treated them with LFcin17–30 orD-LFcin17–30, and
measured M. avium growth after 5 days. Our results, presented in
Fig.3C, showed that, similarly to what was observed before in
BALB/c macrophages (Fig.1D), only D-LFcin17–30 significantly
inhibited the growth of M. avium inside its host cell(Fig. 3C).
Strikingly, the effects of D-LFcin17–30 on M. avium intracellular
growth weresimilar for C57BL/6 and Tnf�/� BMM, leading us to
conclude that TNF-� is not necessaryfor the antibacterial effect of
this peptide. By measuring cytokine levels in
macrophagesupernatants, we confirmed not only that Tnf�/� BMM did
not produce TNF-� but alsothat these macrophages did not produce
significant amounts of IL-6, showing that theeffect of the peptide
is also IL-6 independent (data not shown).
FIG 3 Roles of cytokines in the antimycobacterial activities of
lactoferricin peptides. Twenty-four hours after infection and
treatment with 40 �M LFcin17–30(gray) or D-LFcin17–30 (black), the
levels of IL-6 (A) and TNF-� (B) were determined in the supernatant
of BALB/c BMM. The graphs represent the averages �standard
deviations of results from three independent experiments, presented
as the fold increase relative to their levels in noninfected
control macrophages.Statistical analysis was performed using
one-way ANOVA with Tukey’s multiple-comparison test. *, P � 0.05;
**, P � 0.01; ***, P � 0.001 compared to nontreatedwells. (C) M.
avium 2447 SmT growing inside C57BL/6 and Tnf�/� BMM were treated
with 40 �M LFcin17–30 (gray) or D-LFcin17–30 (black). After 5 days
ofincubation, bacteria were quantified by a CFU assay. The graph
represents the averages from two independent experiments, expressed
as the percentage ofgrowth of mycobacteria in each well relative to
the growth of mycobacteria in the nontreated infected wells
(control) in each experiment. Statistics wereperformed using
two-way ANOVA with Tukey’s multiple-comparison test. **, P � 0.01;
***, P � 0.001 compared to nontreated wells (control).
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D-LFcin17–30 induces ultrastructural alterations on M.
avium-infected macro-phages. To gain an in-depth knowledge of the
mechanisms by which D-LFcin17–30inhibits mycobacterial growth,
transmission electron microscopy (TEM) was performedon M.
avium-infected macrophages treated with the lactoferricin peptides.
Represen-tative images of these assays are shown in Fig. 4.
Striking alterations in macrophageultrastructure were evident when
they were treated with D-LFcin17–30 (Fig. 4C). As wasexpected,
intact mycobacteria were difficult to detect, whereas in nontreated
macro-phages or even in LFcin17–30-treated macrophages, intact
mycobacteria were visual-ized (Fig 4A and B, arrowheads). Several
double-membrane vesicles containing di-gested material, suggestive
of autophagosomes (Fig. 4C, asterisks), were observed
inD-LFcin17–30-treated macrophages. A high number of dense vesicles
and multivesicu-lar bodies loaded with dense material were also
seen (Fig. 4C, black arrows). Largestructures, exhibiting several
membranes and delimitations inside, suggestive of cellmaterial
ingestion and fusion with endosomes, lysosomes, or autophagosomes
(Fig. 4C,white arrows), were frequently seen. These alterations
were not observed in the case ofcells treated with LFcin17–30 (Fig.
4B). These observations suggested that D-LFcin17–30induced
significant alterations in the macrophage vesicular traffic and
membranedigestion pathways, which might contribute to mycobacterial
killing.
M. avium-infected macrophages have increased lysosomal content
and au-tophagic vesicles upon D-LFcin17–30 treatment. Given the
striking morphologicalalterations induced by D-LFcin17–30 on M.
avium-infected macrophages (Fig. 4C) andthe known role of cellular
processes such as apoptosis, autophagy, and lysosomalfusion in the
macrophage-mycobacterium interaction (13, 43), we sought to
quantita-tively evaluate these processes in live-cell experiments.
Macrophages were infectedwith M. avium 2447 SmT and treated with
either LFcin17–30 or D-LFcin17–30. After 4,24, 48, 72, 96, and 120
h of incubation, the cells were analyzed for the three
above-mentioned parameters (Fig. 5). We observed no significant
changes in the levels ofapoptosis or necrosis under any of the
tested conditions, including treatment witheither LFcin17–30 or
D-LFcin17–30 (data not shown). In order to evaluate the levels
ofautophagy, we used the CYTO-ID kit, which is based on a cationic
amphiphilic tracerdye that labels vacuoles associated with the
autophagy pathway and should notaccumulate within lysosomes (44).
When we measured the total fluorescence intensityassociated with
autophagic vesicles, we saw that D-LFcin17–30 slightly but
significantlyincreased the macrophages’ autophagic-vesicle content
(Fig. 5A and B). Regarding theevaluation of the lysosomal content,
macrophages treated with D-LFcin17–30 had a3-fold increase in
density levels of the LYSO dye, which accumulates in live
acidicorganelles, such as lysosomes (Fig. 5C and D). D-LFcin17–30
induced a similar increasein the lysosomal content of noninfected
macrophages (data not shown), indicating thatthis effect is
independent of mycobacterial infection. In agreement with the TEM
results(Fig. 4), large vesicles could be seen in cells treated with
D-LFcin17–30, and these werelabeled with both LYSO and CYTO probes
(Fig. 5B and D), indicating that thesestructures can exhibit both
lysosome and autophagosome features.
FIG 4 Ultrastructural alterations induced by lactoferricin
peptides on M. avium-infected macrophages. Transmission electron
microscopy ofBALB/c BMM infected with M. avium 2447 SmT (A) and
treated with 40 �M LFcin17–30 (B) or D-LFcin17–30 (C) for 5 days.
Scale bar, 2 �m. Symbols:black arrowheads, intact mycobacteria;
black arrows, dense and multivesicular bodies; white arrow, large
dense structures probably resulting frommultivesicular fusion and
digestion; asterisk, double-membrane vesicles.
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DISCUSSION
In this work, we show that lactoferricin-based antimicrobial
peptides strongly inhibitthe growth of M. avium inside its natural
host cell, the macrophage, enhancing theeffect of the conventional
antibiotic ethambutol. Moreover, we show that theD-enantiomer of
lactoferricin, D-LFcin17–30, activates lysosomal and autophagic
path-ways in the macrophage, which can be crucial for its capacity
to kill intracellularmycobacteria.
In a previous work, we showed that LFcin17–30, its variants with
arginines replacedwith lysines and vice versa (LFcin17–30 all K and
LFcin17–30 all R), and its D-enantiomer(D-LFcin17–30) were all
active against M. avium in axenic cultures (40). In contrast, wenow
verify that only D-LFcin17–30 induces a significant decrease in
mycobacterialgrowth inside macrophages. However, all peptides were
effective when combined withthe antibiotic ethambutol. The
combination of antimicrobial peptides with conven-tional
antibiotics is of great potential interest, as it might reduce the
dosages of eachcompound, diminish the probability of resistance,
and reduce the treatment time. Inthe clinics, ethambutol is used in
combination with other antimycobacterial drugs, notonly as a
strategy to prevent the appearance of resistant strains but also
due to its hightoxicity when given alone in high doses (9, 41). The
advantageous combination ofethambutol and iron chelators in the
control of M. avium growth inside macrophageswas recently reported
(45). Ethambutol acts by impairing the biosynthesis of the
cellwall, increasing cell permeability, and potentiating the
actions of other drugs (9, 46, 47).The improvement in the
antimycobacterial activity observed in the present work when
FIG 5 Autophagic and lysosomal content of M. avium-infected
macrophages treated with lactoferricin peptides. At the endof 4,
24, 48, 72, 96, and 120 h of infection with M. avium 2447 SmT and
treatment with D-LFcin17–30, macrophages wereincubated with CYTO-ID
(A, B) or LYSO-ID (C, D) detection kits for 30 min at 37°C. (A, C)
The results represent the averages fromthree independent
experiments, expressed as the fold increase in the fluorescence
level of each detection reagent under eachcondition and time point
relative to the fluorescence level of the noninfected nontreated
well at 4 h. Statistics were performedusing two-way ANOVA with
Tukey’s multiple-comparison test. **, P � 0.01; ***, P � 0.001
compared to nontreated infectedwells. White circles, M.
avium-infected macrophages; gray squares, M. avium-infected and
LFcin17–30-treated macrophages;black squares, M. avium-infected and
D-LFcin17–30-treated macrophages. (B, D) Representative pictures of
one experiment outof three of M. avium-infected macrophages (top)
and treated with D-LFcin17–30 (bottom) at 4, 72, and 120 h with
CYTO-ID (B)or LYSO-ID (D). Scale bar, 10 �m.
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ethambutol was administered together with the peptides is
probably related to in-creased membrane permeability induced by
either ethambutol, the peptides, or both,allowing for higher
concentrations of the compounds to enter the cell and
potentiatingtheir mutual activities.
Given that D-LFcin17–30 was more active than the L-peptides, we
proceeded toinvestigate the mechanism by which the D-enantiomer of
LFcin17–30 exerts its anti-mycobacterial activity inside
macrophages. Due to their peptidic nature, AMP are
highlysusceptible to proteases and other plasma components. This
feature is one of theobstacles to AMP application in the clinics,
as it results in low stability and bioavail-ability, limiting most
current AMP applications to topical agents (48). One
strategyemployed to overcome this problem is the use of nonnatural
D-enantiomers of aminoacids, as they are more resistant to
proteolytic activity (49). In fact, several reportsdescribe that
AMP, including lactoferricin derivatives, composed of D-amino acids
aremore resistant to degradation and have activities higher than or
similar to those of theircounterparts with L-amino acids (50–56).
In the case of the peptides studied in thiswork, D-LFcin17–30 was
capable of resisting degradation, and it persisted in cell
culturemedium at a higher concentration than LFcin17–30 over time,
indicating that this isprobably a crucial factor for its higher
antimycobacterial activity. The fact that theD-enantiomer is more
active than the L-enantiomer also reveals that the
observedantimicrobial effect is probably not related to chiral
receptors, because they would notrecognize D-amino acids.
Because we had reported previously that these peptides exhibit a
direct antimicro-bial effect against M. avium in broth culture
(40), we initially hypothesized that theinhibition of mycobacterial
growth inside macrophages is the result of a direct effect onthe
mycobacteria. However, when we studied the distribution and
subcellular localiza-tion of LFcin17–30 and D-LFcin17–30 inside M.
avium-infected macrophages, we saw nocolocalization between AMP and
bacteria. We performed all the assays in live cells
withfluorochrome-labeled peptides to avoid fixation-related
artifacts, but we failed todetect any colocalization, even in the
presence of ethambutol, at any incubation time.The peptides seemed
to follow an endocytic pathway, colocalizing with
fluorescein-conjugated dextran. Although we cannot exclude a
possible alteration in peptidedistribution caused by the
fluorochrome link, the additional assays performed clearlyindicate
that D-LFcin17–30 impacts macrophage biology, and this may cause
mycobac-terial killing rather than having a direct action on the
bacteria.
The administration of LFcin17–30 and D-LFcin17–30 was
accompanied by increasedlevels of TNF-� and IL-6 production by M.
avium-infected macrophages. TNF-� isnecessary for the hosts’
resistance to M. avium. This cytokine is involved in
macrophageactivation, being able to induce intracellular killing of
mycobacteria (19, 20, 57, 58). Inturn, IL-6 is a cytokine involved
in the modulation of inflammation and the acute-phaseresponse,
important for host responses to mycobacterial infections (59).
Although bothpeptides increased the levels of TNF-� and IL-6, these
are not essential for theantimicrobial effect of D-LFcin17–30, as
their absence did not interfere with thepeptide’s effect. We did
not detect increased production of nitrite in D-LFcin17–30-treated
macrophages (data not shown). Furthermore, we did not expect nitric
oxide tobe involved in the antimycobacterial effect of
D-LFcin17–30, since we have previouslyshown that oxygen- and
nitrogen-reactive species are not important for the control ofM.
avium growth inside murine macrophages (24, 25).
Lactoferricin has been reported to have multiple roles in the
host immune response.Besides having a direct antimicrobial activity
on several pathogens, lactoferricin caninhibit septic shock by
binding to endotoxins (60). Additionally, it has been shown
toselectively kill cancer cells (61–66) in a process involving both
apoptosis and autophagy(66). Autophagy is a host cell effector
mechanism used as a quality control for theremoval of protein
aggregates and damaged organelles. Under stress conditions, thecell
can activate autophagy for survival, selectively targeting
different cargos fordegradation. Xenophagy, the autophagic
degradation of intracellular pathogens, is aninnate defense weapon
used by a host to control pathogen replication and prolifera-
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tion (67). In the case of mycobacterial infections, vitamin D3
concomitantly induces theproduction of antimicrobial peptides (such
as cathelicidin) and autophagy, both ofwhich play a role in the
control of the pathogen’s growth within macrophages
(32–34).Interestingly, the peptide Beclin-1 was shown to control
mycobacterial growth insidemacrophages by inducing autophagy (68),
and the D-form of the peptide induceshigher activation of this
pathway (69).
The ultrastructural changes observed in this work when M.
avium-infected macro-phages were treated with D-LFcin17–30,
together with the increase in lysosomal andautophagic vesicles,
lead us to conclude that the peptide facilitates the targeting
ofmycobacteria to lysosomal degradation. Given that D-LFcin17–30 is
composed ofnonnatural D-amino acids, the cells may recognize the
peptide as a stress signal, leadingto downstream activation of
inflammatory pathways. We cannot clearly distinguishwhether
autophagy or phagosomal maturation is being activated. These two
pathwaysoverlap and can have common denominators (e.g., human VPS34
and RAB7) (8, 67, 70).Either way, we postulate that lactoferricin
primes mycobacteria for vesicular digestion,having phagosomes or
autophagosomes fusing with lysosomes for cargo degradation.
In summary, in this work, we showed that a D-enantiomer of
lactoferricin, D-LFcin17–30, modulates macrophage activity toward a
state which favors mycobacterial elimi-nation. This observation,
together with the data on the safe use of lactoferricin peptidesto
improve animal health in different mouse models (64, 71, 72), opens
the way towarda possible use of this peptide to treat mycobacterial
infections as an adjunct therapywith conventional antibiotics.
Additionally, these data suggest other possible applica-tions for
D-LFcin17–30 in situations requiring macrophage activation.
MATERIALS AND METHODSPeptides. Bovine lactoferricin peptides
(LFcin17–30, D-LFcin17–30, LFcin17–30 all K, and LFcin17–30 all
R)
(Table 1) were synthesized by solid-phase peptide synthesis
using 9-fluorenyl-methoxycarbonyl (Fmoc)chemistry with a Syro II
synthesizer (Biotage, Uppsala, Sweden) as described previously
(73). Peptidesynthesis-grade solvents were obtained from Actu-All
Chemicals (Oss, The Netherlands), the preloadedNovaSyn TGA resins
from Novabiochem (Merck Schuchardt, Hohenbrunn, Germany), and the
N-�-Fmoc-amino acids from ORPEGEN Pharma (Heidelberg, Germany) and
Iris Biotech (Marktredwitz, Germany).LFcin17–30 and D-LFcin17–30
were labeled in synthesis with 5(6)-carboxytetramethylrhodamine
(TAMRA;Novabiochem) by coupling TAMRA to the �-amino group of an
additional C-terminal lysine residue usingFmoc-Lys(ivDde)-OH,
resulting in a labeling stoichiometry of 1:1, without any free
TAMRA remaining. Briefly,the peptide was synthesized as described
above on
N-�-Fmoc-N-�-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine
coupled to NovaSyn TGR resin (Novabiochem) with the N-terminal
amino acidprotected by N-�-tert-butoxycarbonyl. Subsequently, the
ivDde-protecting group at the C-terminal Lys wasreleased by
hydrazinolysis (2% hydrazine hydrate in N-methyl-2-pyrrolidone
[NMP]) followed by overnightincubation with 1.5 eq TAMRA in (NMP)
containing 1.5 eq of 1-hydroxybenzotriazole (HOBt), 1.7 eq
of2-1[H-benzotriazole-1-yl]-1,1,3,3-tetramethylaminium
tetrafluoroborate (TBTU), and 70 �l of N,N-diiso-propylethylamine
(DIPEA) in a final volume of 2 ml. Next, the peptide-containing
resin was washed twice withNMP and twice with 20% piperidine,
followed by three consecutive washes with NMP, isopropyl alcohol
(IPA),and dichloromethane (DCM). Subsequently, the peptide was
detached from the resin and deprotected asdescribed previously
(73).
Peptides were purified to a purity of at least 95% by
semipreparative reverse-phase HPLC (RP-HPLC)(JASCO Corporation,
Tokyo, Japan) on a Vydac C18 column (catalog number 218MS510;
Vydac, Hesperia,CA, USA), and the authenticity of the peptides was
confirmed by matrix-assisted laser detectionionization–time of
flight (MALDI-TOF) mass spectrometry on a Microflex LRF mass
spectrometerequipped with an additional gridless reflectron (Bruker
Daltonik, Bremen, Germany) as describedpreviously (73).
All purified peptides were freeze-dried. Peptide stock solutions
were prepared in phosphate-bufferedsaline (PBS; pH � 7.4), with 10%
dimethyl sulfoxide (DMSO) in the case of the labeled peptides,
andstored at �20°C until use.
HPLC. Peptides (LFcin17–30 and D-LFcin17–30) were incubated with
Dulbecco’s modified Eagle’smedium (DMEM), supplemented as stated
below, at 37°C for 4 days. At the end of 0, 0.5, 2, 4, 8, 24,
48,72, and 96 h, an aliquot was taken from each mixture and
analyzed by high-performance liquidchromatography (HPLC). The HPLC
(Hitachi Elite Autosampler L-2200, pump L-2130, diode array
detectorL-2455, and column oven L-2300) was performed with a
150-mm-diameter C18 reverse-phase column(Merck). Each analysis
involved an injection volume of 40 �l and elution with 0 to 100%
acetonitrile in0.05% aqueous trifluoroacetic acid (TFA) at a flow
rate of 1 ml/min; the detection wavelength was set to220 nm. The
chromatograms were analyzed with EZChrom Elite software, and the
peaks were integratedto extract the area.
Bacteria. In this work, two strains of Mycobacterium avium were
used: (i) M. avium strain 2447 smoothtransparent variant (SmT),
originally isolated by F. Portaels (Institute of Tropical Medicine,
Antwerp,
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Belgium) from an AIDS patient, and (ii) M. avium 104:pMV306
(hsp60 gfp) expressing green fluorescentprotein (M. avium-GFP)
(74). Mycobacteria were grown and stored as described previously
(40).
BMM. Macrophages were derived from the bone marrow of male
BALB/c, C57BL/6, and C57BL/6TNF-�-deficient (Tnf�/�) mice bred at
the i3S/IBMC animal facility. TNF-�-deficient breeder mice
wereoriginally purchased from B & K Universal (East Yorkshire,
UK). Bone marrow-derived macrophages (BMM)were obtained as
described previously (75).
Macrophage infection and quantification of bacterial growth. BMM
at day 10 of culture wereinfected with 106 CFU of M. avium 2447 SmT
for 4 h at 37°C in a 7% CO2 atmosphere. After incubation,cells were
washed several times to remove noninternalized bacteria and
reincubated with new mediumwith or without 40 �M peptide (76.9
�g/ml LFcin17–30 and D-LFcin17–30, 73.6 �g/ml LFcin17–30 all R,80.3
�g/ml LFcin17–30 all R), alone or in combination with the
antibiotic ethambutol (2 �g/ml or 7.2 �Methambutol dihydrochloride;
Sigma-Aldrich, St. Louis, MO, USA). Each condition was tested in
triplicate.After 5 days in culture, the intracellular growth of M.
avium 2447 SmT was evaluated by determining thenumber of CFU, as
described previously (75).
Measurement of macrophage viability. The viability of BALB/c BMM
was determined by resazurinreduction. After 24 h of infection and
peptide treatment, the supernatant was removed and macro-phages
were incubated with new medium containing 125 �M resazurin
(Sigma-Aldrich, St. Louis, MO,USA) for 24 h at 37°C in a 7% CO2
atmosphere. The fluorescence of resorufin, resulting from
theconversion from resazurin by metabolically active cells, was
measured at an excitation wavelength (�ex)of 560 nm and an emission
wavelength (�em) of 590 nm.
Peptide’s distribution and localization inside macrophages.
BALB/c BMM were cultured in �-Slide8-well plates (ibidi GmbH,
Germany). At the 10th day of culture, macrophages were infected
with eitherM. avium-GFP or M. avium 2447 SmT and treated with 10 �M
LFcin17–30 —TAMRA or D-LFcin17–30 —TAMRA. Simultaneously, half of
the M. avium-GFP-infected macrophages were treated with 7.2
�Methambutol. Fluorescein-conjugated dextran (molecular weight,
10,000) (22.5 �M, final concentration)(Molecular Probes,
Invitrogen, Carlsbad, CA, USA) or MitoTracker Green FM (200 nM,
final concentration)(Molecular Probes, Invitrogen, Carlsbad, CA,
USA) was added to M. avium 2447 SmT-infected macro-phages for
endosomal or mitochondrial labeling, respectively.
Fluorescein-conjugated dextran wasadded along with the peptides
immediately after infection and incubated for 2 h, whereas
MitoTrackerGreen FM was incubated for 30 min prior to
visualization. Macrophages were observed and photo-graphed live,
using a Leica TCS SP5II laser scanning confocal microscope (Laser
Microsystems, Germany)with a 63� oil objective. Immediately before
visualization, cells were washed with PBS and kept in RPMImedium
without phenol red (Life Technologies, Inc., Paisley, UK).
Cytokine production. Cytokine production was evaluated in the
supernatants of macrophagecultures 24 h after infection with M.
avium 2447 SmT and peptide treatment. The levels of six
differentcytokines (IL-12p70, TNF-�, IFN-�, CCL2, IL-10, and IL-6)
were determined using the BD cytometric beadarray (CBA) mouse
inflammation kit (BD Biosciences, San Jose, CA, USA) according to
the manufacturer’sinstructions. Briefly, standards and samples were
incubated for 2 h with a mixture of capture beads foreach cytokine
and with a mixture of phycoerythrin (PE)-conjugated antibodies as a
detection reagent.Afterward, the wells were washed, the supernatant
was discarded, and the beads were resuspended inwash buffer. The
standards and samples were then acquired in a BD FACSCanto II
cytometer (BDBiosciences, San Jose, CA, USA) and the results
analyzed using the FCAP Array software (BD Biosciences,San Jose,
CA, USA).
Transmission electron microscopy. In brief, BALB/c BMM infected
with M. avium 2447 SmT andtreated with lactoferricin peptides for 5
days were fixed with 2.5% glutaraldehyde (Electron
MicroscopySciences, Hatfield, PA, USA) and 2% paraformaldehyde
(Merck, Darmstadt, Germany) in cacodylate buffer(0.1 M, pH 7.4) for
2 h at room temperature. Samples were dehydrated and embedded in
Epon resin(TAAB, Berks, England). Ultrathin sections (40- to 60-nm
thickness) were prepared on an RMC Ultrami-crotome (Powertome, USA)
using diamond knives (DDK, Wilmington, DE, USA). The sections
weremounted on 200-mesh copper or nickel grids, stained with uranyl
acetate and lead citrate for 5 min each,and examined under a JEOL
JEM 1400 TEM (Tokyo, Japan). Images were digitally recorded using
an Oriuscharge-coupled-device (CCD) digital camera (1,100 W; Gatan,
Tokyo, Japan) at the HEMS/i3S of Univer-sidade do Porto, Porto,
Portugal.
Live-cell imaging. For live-cell imaging, BALB/c BMM were
cultured on �-Plate 96-well ibiTreat (ibidiGmbH, Germany) as stated
above. After M. avium infection and treatment with lactoferricin
peptides(time zero), the cells were incubated at 37°C in a 7% CO2
atmosphere, and at the end of 4, 24, 48, 72, 96,and 120 h, the
levels of apoptosis and necrosis, the lysosomal content, and the
autophagic levels wereassessed separately. For that, Enzo
Cellestial fluorescent probes were used from the
apoptosis/necrosisdetection kit, LYSO-ID detection kit, and CYTO-ID
autophagy detection kit (Enzo Life Sciences Inc., USA).According to
the manufacturer’s instructions, at each time point the cells were
washed and incubatedwith the respective detection reagents for 30
min at 37°C in a 7% CO2 atmosphere. For visualization andimage
acquisition, macrophages were incubated with PBS-5% fetal bovine
serum (FBS). Images werecollected in a controlled environment (37°C
and CO2 atmosphere) with a Nikon 40�/0.95-numerical-aperture (NA)
Plan Fluor objective in a high-throughput automated fluorescence
wide-field microscope(IN cell analyzer 2000; GE Healthcare, Little
Chalfont, UK). The 2.5-dimensional (2.5-D) acquisition
anddeconvolution mode was used to integrate the signal over a
1.5-�m Z-section, generating a pseudo 3-Dprojection. Each well was
screened for 10,000 nuclei (up to 72 fields). Quantification of the
fluorescencelevels (expressed as the mean density value of the
pixels) of each detection reagent (apoptosis, necrosis,LYSO-ID, and
CYTO-ID detection reagents) was performed with Developer Toolbox
1.9.2 (GE Health-care, Little Chalfont, UK). Briefly, nuclear and
cytoplasm segmentation algorithms were used to
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identify and quantify the number of cells under all conditions.
The fluorescence level of eachindividual cell, under each condition
and kit, was measured, allowing us to calculate the
meanfluorescence value for each well.
Statistical analysis. Statistical analyses were performed with
GraphPad Prism 6 (GraphPad Software,Inc., La Jolla, CA, USA) using
two-way analysis of variance (ANOVA) with Tukey’s
multiple-comparison test.Differences with a P value under 0.05 were
considered significant (*, P � 0.05; **, P � 0.01; ***, P �
0.001).
ACKNOWLEDGMENTSWe thank the valuable collaboration of the
following scientific services at i3S: the
Advanced Light Microscopy Unit (ALM), Histology and Electron
Microscopy Service(HEMS), BioSciences Screening Unit (BSU), and
Animal Facility. We also thank JoãoRelvas and Renato Socodato from
the Glial Cell Biology group of IBMC/i3S, Universidadedo Porto,
Porto, Portugal, for kindly providing the C57BL/6 TNF-�-deficient
mice.
We declare that we have no competing interests.T.S., J.G.M.B.,
M.B., and M.S.G. designed the research; T.S., A.C.M., K.N., T.M.,
and N.V.
performed research; M.R, P.G., J.G.M.B., and M.B. contributed
with reagents and analytictools; T.S., A.C.M., M.B., J.G.M.B.,
P.N.R., and M.S.G. analyzed the data; and T.S. and M.S.G.wrote the
paper with contributions from A.C.M., T.M., N.V., M.R., P.G., K.N.,
J.G.M.B.,P.N.R., and M.B.
This research received funding support from the Fundação Para a
Ciência e Tecno-logia, European Social Funds, Programa Operacional
Regional do Norte (ON.2–O NovoNorte), under the Quadro de
Referência Estratégico Nacional (QREN), the Fundo Euro-peu de
Desenvolvimento Regional (Feder), and the Programa Operacional da
Competi-tividade e Internacionalização (POCI) under COMPETE 2020
(grant SFRH/BD/77564/2011to T.S.; grant SFRH/BPD/101405/2014 to
A.C.M.; grant SFRH/BD/79874/2011 to T.M.;grant IF/00092/2014 to
N.V.; grant PTDC/IMI-MIC/1683/2014 to M.S.G.; grant
UID/MULTI/04378/2013 POCI-01-0145-FEDER-007728 to M.R., P.G., and
N.V.; grant UID/QUI/0081/2013 POCI-01-0145-FEDER-006980 to M.B.;
grant NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions to
P.N.R.; grant NORTE-07-0162-FEDER-000111 to P.G.;grant
NORTE-07-0124-FEDER-000066 to M.R.; and grant
NORTE-01-0145-FEDER-000024-DESignBIOtechHealth to P.G.). This work
also benefited from a grant from theUniversity of Amsterdam for
research into the focal point Oral Infections and Inflam-mation,
given to J.G.M.B. and K.N.
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RESULTSUp to 40 µM, lactoferricin peptides are not toxic to
primary mouse macrophages. Lactoferricin peptides inhibit M. avium
growth inside macrophages and synergize with ethambutol.
D-LFcin17–30 is more resistant to degradation by medium components
than LFcin17–30. Lactoferricin peptides do not colocalize with M.
avium inside macrophages. Lactoferricin peptides increase
macrophage production of proinflammatory cytokines. The
antimicrobial effects of lactoferricin peptides inside macrophages
are not dependent on the production of TNF- and/or of IL-6 by
macrophages. D-LFcin17–30 induces ultrastructural alterations on M.
avium-infected macrophages. M. avium-infected macrophages have
increased lysosomal content and autophagic vesicles upon
D-LFcin17–30 treatment.
DISCUSSIONMATERIALS AND METHODSPeptides. HPLC. Bacteria. BMM.
Macrophage infection and quantification of bacterial growth.
Measurement of macrophage viability. Peptide’s distribution and
localization inside macrophages. Cytokine production. Transmission
electron microscopy. Live-cell imaging. Statistical analysis.
ACKNOWLEDGMENTSREFERENCES